The RNA exosome is a RNA degradation machinery that plays a critical role in RNA quality control by degrading erroneous or incorrectly spliced RNAs. In many cancers, such as pancreatic cancer, the splicing process is compromised, resulting in the generation of large amounts of aberrant transcripts. We hypothesize that cancer cells are uniquely dependent on RNA exosome activity to remove excess amounts of aberrant transcripts and that the RNA exosome therefore may be an impactful new target for therapeutic intervention for pancreatic cancer. We have developed a set of first-in-class small molecule inhibitors of the RNA exosome and will in this proposal refine these compounds and assess their tumor- targeting efficacy for pancreatic cancer in three specific Aims. In Aim 1 a lead optimization campaign will be initiated of our first-in-class RNA exosome inhibitors. The refined compounds will be evaluated for their RNA exosome-targeting activities using both in vitro and cell-based RNA exosome activity assays and we will identify their cellular targets. In Aim 2, the potencies of the 10 top RNA exosome inhibitors will be assessed by MTT and clonogenic survival assays to select the top three compounds for in vivo studies. These top three compounds will then be used in combination with either ionizing radiation or gemcitabine to assess synergy. Inducible shRNA and auxin-inducible protein degradation technologies will be used to inactivate the RNA exosome and these systems will be compared with the top three inhibitors to explore fundamental mechanisms of action of the RNA exosome in transcriptional and post-transcriptional events. In Aim 3, we will test the 3 most promising compounds in a PDX orthotopic model for pancreatic cancer either alone or in combination regimens with gemcitabine and nab-paclitaxel. This is a very innovative proposal exploring a novel cancer therapeutic target and highly focused on the development of new impactful treatments for pancreatic cancer. Upon the successful completion of this grant period, we intend to perform IND-enabling studies of the best candidate compounds.